We plan to continue studying mitochondrial DNA, its replication, and the enzymology of replication: 1. Enzymology: (a) Nicking-closing enzyme - We have now sufficiently purified this enzyme from rat liver mitochondria to study its properties, to seek confirmation of our preliminary results showing this and the nuclear enzyme to be distinct, and to study its possible role in mtDNA replication at the enzymatic level. (b) DNA gyrase - We have not yet detected this enzyme in either rat liver or mouse L-cell mitochondria but the presence of the nicking-closing enzyme, a subunit of two bacterial gyrases, spurs us to continue the search. We plan to study its possible role in mtDNA replication, and its structural and functional relationship to the nicking-closing enzyme. 2. Polymorphism of mitochondrial DNA - Using restriction endonucleases, we have found two types of mtDNA distributed throughout the population of the laboratory rat (a single rat possessing a single type). We plan to study (a) the nature of the structural differences between the two types including a comparison of the restriction enzyme cleavage maps as well as the physical genomic maps; and (b) the problem of what, if any, phenotypic differences exist between the two types of DNA. 3. Dense DNA - We have isolated a new DNA-containing structure from rat liver mitochondria. Its importance in replication is suggested by its high rate of incorporation of DNA precursors. Its high buoyant density (1.760 g/ml) suggests the presence of RNA. Electron microscopy shows a complex structure consisting of a hazy core containing some electron dense regions and a multiplicity of loops emanating from the core. We plan to investigate the composition, structure, origin and role of this material.